1. Introduction

The genus Melaleuca (family Myrtaceae, subfamily Leptospermoideae) consists of about 230 species rich in volatile oils. Several studies demonstrated the efficacy of some Melaleuca essential oils against different types of bacteria and fungi [1–4], as in the case of Melaleuca alternifolia essential oil against methicillin-resistant Staphylococcus aureus (MRSA), Legionella pneumophila, and Staphylococcus aureus[5].

Essential oils are becoming more popular, because many synthetic drugs are connected with unpleasant side effects, such as nephrotoxicity or ototoxicity. Volatile oils also represent an interesting alternative due to an emerging resistance of microorganisms against synthetic agents. Essential oils can exert not only bacteriostatic and bactericidal effects, but also demonstrate activity against fungi and yeasts [5].

Moreover, in the past, it seemed that certain Melaleuca species may have allelopathic properties, resulting in an inhibition of other species in the same ecosystem [6]. The bare ground in Melaleuca forests was reported as an example of allelopathy in this genus [7].

Melaleuca armillaris Sm. is one of the most widely cultivated melaleucas. It is commonly known as Bracelet Honey Myrtle and grows into a large spreading shrub or small tree. Literature reports about M. armillaris remain scarce. GC/MS investigations of its essential oil revealed the presence of 1,8-cineole as the main component [8–12]. Only little information could be found in the literature about the composition of Melaleuca styphelioides Sm. oils. Farag and coworkers [12] reported that the essential oil of this species contained mainly caryophyllene oxide (43.8%) and (−)-spathulenol (9.7%). Previously, the same authors [13] reported that the essential oil of M. styphelioides contained mainly caryophyllene (49.9%) and methyl eugenol (26.6%). Only a few reports are available about Melaleuca acuminata F. Muell. [14,15].

In continuation of our studies on the possible phytotoxic and antimicrobial activity of essential oils from plants collected in the Mediterranean area [16,17], we studied the chemical composition of the essential oils from Melaleuca armillaris, M. acuminata and M. styphelioides and their possible in vitro effects against germination and initial radicle elongation of Raphanus sativus L. (radish), Lepidium sativum L. (garden cress), Sinapis arvensis L. (wild mustard), Triticum durum L. (wheat) and Phalaris canariensis L. (canary grass) and the antimicrobial activity against eight selected microorganism.

2. Results and Discussion

2.1. Chemical Composition of the Essential Oils

Hydrodistillation yielded 0.65%, 0.53% and 0.35% of essential oil (on a dry mass basis) for M. armillaris, M. acuminata and M. stypheliodes, respectively. Table 1 shows the chemical composition of the three Melaleuca oils; compounds are listed according to their elution order on a HP-5MS column. In all, 46 compounds were identified, 38 for M. armillaris, accounting for 99.3% of the total oil, 20 for M. acuminata, accounting for 99.7% of the total oil, and eight for M. styphelioides (92.4%), respectively.

In the oil from M. armillaris, the oxygenated monoterpenoids amounted to 44.6%; on the other hand, the total sesquiterpenic fraction amounted to 51.6% of the total oil. The main compounds are the sesquiterpene cis-calamenene (19.0%) and the oxygenated sesquiterpene torreyol (15.1%). Other compounds, in a lesser amount, are dihydrocarveol (9.0%) and α-terpineol (7.7%), both oxygenated monoterpenoids. In the literature, Chabir and coworkers [8,9] reported 1,8-cineole (85.8%) as the most abundant compound in M. armillaris. The major compound in the essential oil of M. styphelioides was methyl eugenol (91.1%), a phenolic compound. In the literature, Farag and coworkers [12] reported that the essential oil of this species contained mainly caryophyllene oxide (43.8%), followed by (−)-spathulenol (9.7%). In another paper [13], the same authors reported that the essential oil of this species contained mainly caryophyllene (50.0%) and methyl eugenol (26.6%).

In the oil from M. acuminata, the oxygenated monoterpenoids amounted to 95.6%, with a total sesquiterpenes amount of 1.7% (0.6% sesquiterpene hydrocarbons and 1.1% of oxygenated sesquiterpenes) of the total oil. trans-Pinocarveol (25.1%), dihydrocarveol (23.6%), myrtenol (12.3%) and 1,8-cineole (11.7%) were the most abundant among the oxygenated monoterpenes. In the literature, only two references reported the chemical composition of the essential oil of M. acuminata: Smith and coworkers [14,15] reported that cineole was the main component of the essential oil of M. acuminata.

2.2. Phytotoxic Activity

The three essential oils were evaluated for their phytotoxic activity against germination and radicle elongation (Table 2) of radishes and garden cress, two species frequently utilized in biological assays, and of wild mustard, wheat and canary grass, three weed species.

The oils seem to be ineffective against germination, but they affected the radicle elongation of the five tested seeds. The essential oil of M. styphelioides, at all doses tested, significantly inhibited the radicle elongation of garden cress. The radicle elongation of wild mustard and radish were inhibited by M. acuminata oil at the highest doses (2.5 μg/mL, 1.25 μg/mL) used. At doses of 1.25 and 0.625 μg/mL, the essential oil of M. acuminata significantly inhibited the radicle elongation of canary grass (Table 2). The difference in biological activity of the oils could be attributed to their different chemical composition.

On the other hand, the oil of M. acuminata was rich in oxygenated monoterpenoids, trans-pinocarveol, dihydrocarveol, myrtenol and 1,8-cineole. trans-Pinocarveol was reported as one of the main components of the strong phytotoxic oil from a Cistus ladanifer L. population [18].

Yatagai and coworkers [19] reported that the leaf oil of Melelauca bracteata F. Muell. had the strongest germination and growth-inhibition activity against radish seeds.

The roots were probably more sensitive than shoots to the phytotoxic activity of the oil; the process of germination was active while the oil probably affected the elongation process. Such activity of the essential oils could help to explain the ecological role of the genus Melaleuca in the Mediterranean area.

2.3. Antimicrobial Activity

The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) values of the essential oils against eight selected microorganisms are reported in Table 3.

The essential oils showed inhibitory activity against the gram-positive pathogens, among which is S. epidermidis. Among gram-negative bacteria, E. coli was affected by the oil of M. armillaris and M. acuminata. The essential oil of M. acuminata was more active than other oils and presumably this activity is related to the high amounts of oxygenated monoterpenoids.

Farag and coworkers [12,13] reported the effect of the essential oil from the leaves of M. armillaris, M. ericifolia (Smith), M. leucadendron (Linn.) and M. styphelioides against the growth of some microorganisms. The results demonstrated that the degree of the microbial inhibition is largely dependent on the species. M. ericifolia exhibited the highest inhibitory effects against Bacillus subtilis and Aspergillus niger. The antimicrobial properties of Melaleuca essential oil have been reported in several studies [20] Terpinen-4-ol is considered to be the principal active component of Melaleuca alternifolia Cheel (tea tree) oil [21–23]. Terpinen-4-ol could constitute an interesting alternative in the therapy of MRSA infections of the skin [5]. In the literature, it was reported that some members of the Myrtaceae family (Eucalyptus globulus and Melaleuca alternifolia) whose essential oil consisted mainly of monocyclic and bicyclic monoterpenes (e.g., 1,8-cineole and terpinen-4-ol) effectively inhibited the growth of drug-resistant bacterium strains [5].

3. Experimental Section

3.1. Plant Material

Leaves of Melaleuca armillaris, M. acuminata and M. styphelioides Sm. were collected from the Botanical Garden of the National Institute of Researches on Rural Engineering, Water and Forests (Ariana, Tunisia) in April 2011. Five samples were collected from more than five different trees, mixed for homogenization and used in three replicates for essential oil extractions. Specimens were identified by Dr. H. Lamia, and voucher specimens were deposited at the herbarium of the Laboratory of Forestry Ecology at the National Institute of Research on Rural Engineering, Water and Forest (Tunisia).

3.2. Isolation of the Volatile Oils

One hundred grams of dried leaves of each Melaleuca species were ground in a Waring blender and then, subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia[24].

The oils were solubilized in n-hexane, dried over anhydrous sodium sulphate and dried under N2 to remove hexane. Samples were stored at +4 °C in the dark until tested and analyzed.

3.5. Identification of the Essential Oils Components

Most constituents were identified by gas chromatography by comparison of their Kovats retention indices (Ri) with either those of the literature [25,26] or with those of authentic compounds available in our laboratories. The Kovats retention indices were determined in relation to a homologous series of n-alkanes (C10–C35) under the same operating conditions. Further identification was made by comparison of their mass spectra on both columns with either those stored in NIST 02 and Wiley 275 libraries or with mass spectra from the literature [25,27] and a homemade library. The components relative concentrations were obtained by peak area normalization. No response factors were calculated.

3.6. Biological Assay

A bioassay based on germination and subsequent radicle growth was used to study the phytotoxic effects of the three essential oils on seeds of Raphanus sativus L. cv. “Saxa” (radish), Lepidium sativum L. (garden cress) and the three weed species Sinapis arvensis L. (wild mustard), Triticum durum L. (wheat) and Phalaris canariensis L. (canary grass). The seeds of radish and garden cress were purchased from Blumen srl (Piacenza, Italy), while mustard, wheat and canary grass were collected from wild plants. The seeds were surface sterilized in 95% ethanol for 15 s and sown in Petri dishes (Ø = 90 mm) containing five layers of Whatman filter paper impregnated with distilled water (7 mL, control) or a tested solution of the essential oil (7 mL) at the different assayed doses. The germination conditions were 20 ± 1 °C with a natural photoperiod. The essential oils, in a water–acetone mixture (99.5:0.5), were assayed at the doses of 2.5, 1.25, 0.625, 0.25, 0.125 and 0.062 μg/mL. Controls performed with water-acetone mixture alone showed no appreciable differences in comparison with controls in water alone. Seed germination was observed directly in Petri dishes, each 24 h. A seed was considered germinated when the protrusion of the root became evident [28]. After 120 h (on the fifth day), the effects on radicle elongation were measured in centimeter. Each determination was repeated three times, using Petri dishes containing 10 seeds each. Data are expressed as the mean ± SD for both germination and radicle elongation. Data were ordered in homogeneous sets, and the Student’s t test of independence was applied [29].

The antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) using the broth dilution method [30–32]. Eight bacterial species, selected as representative of the gram-positive and gram-negative classes, were tested: Staphylococcus aureus (ATTC 25923), Streptococcus faecalis (ATTC 29212), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis (ATCC 12228), Klebsiella pneumoniae (ATCC 10031) and Proteus vulgaris (ATCC 13315). The strains were maintained on Tryptone Soya agar (Oxoid, Milan, Italy); for the antimicrobial tests, Tryptone Soya broth (Oxoid, Milan, Italy) was used. In order to facilitate the dispersion of the oil in the aqueous nutrient medium, it was diluted with Tween 20 at a concentration of 10%. Each strain was tested with a sample that was serially diluted in broth to obtain concentrations ranging from 100 μg/mL to 0.8 μg/mL. The sample was previously sterilized with a 0.20 μm Millipore filter. The sample was stirred, inoculated with 50 μL of physiological solution containing 5 × 106 microbial cells and incubated for 24 h at 37 °C. The MIC value was determined as the lowest concentration of the sample that did not permit any visible growth of the tested microorganism after incubation. The control, containing only Tween 20 instead of the essential oil, was not toxic to the microorganisms. Cultures, containing only sterile physiologic solution Tris buffer, were used as positive control. MBC was determined by subculture of the tubes with inhibition in 5 mL of sterile nutrient broth. After incubation at 37 °C, the tubes were observed. When the germs did not grow, the sample denoted a bactericidal action. Oil samples were tested in triplicate and the experiment was performed three times. The results are expressed as mean ± SD. Chloramphenicol was used as reference drug [17].

4. Conclusions

Data obtained in this paper could be useful in the chemotaxonomic knowledge of the genus Melaleuca that consists of about 230 species, the chemistry of M. armillaris, M. styphelioides and M. acuminata being little known. Data on phytotoxic activities could help to explain the ecological role of genus Melaleuca in the Mediterranean area. The antimicrobial activity of the essential oil is in agreement with the uses of other Melaleuca species as antimicronial drugs.

Table 3.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Values (μg/mL) of essential oils from the three Melaleuca Species and MIC of the reference antibiotic, chloramphenicol. Results are the mean of three experiments.

Table 3.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Values (μg/mL) of essential oils from the three Melaleuca Species and MIC of the reference antibiotic, chloramphenicol. Results are the mean of three experiments.